Molecules 2011, 16, 3338-3344; doi:10.

3390/molecules16043338

OPEN ACCESS

molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article

Kaempferol and Kaempferol Rhamnosides with Depigmenting

and Anti-Inflammatory Properties

Ho Sik Rho 1,*, Amal Kumar Ghimeray 2, Dae Sung Yoo 2, Soo Mi Ahn 3, Sun Sang Kwon 4,
Keun Ha Lee 4, Dong Ha Cho 2 and Jae Youl Cho 5,*

1
R & D Center, AmorePacific Corporation, Yongin, Korea
2
College of Biomedical Science, Kangwon National University, Chuncheon, Korea
3
Skin Biotechnology Center, Kyung Hee University, Suwon, Korea
4
R & D Center, Morechem Corporation, Seoul, Korea
5
Department of Genetic Engineering, Sungkyunkwan University, Suwon, Korea

* Authors to whom correspondence should be addressed; E-Mails: thiocarbon@freechal.com

(H.-S.R.); jaecho@skku.edu (J.-Y.C.)

Received: 24 March 2011; in revised form: 14 April 2011 / Accepted: 15 April 2011 /
Published: 18 April 2011

Abstract: The objective of this study was to examine the biological activity of kaempferol
and its rhamnosides. We isolated kaempferol (1), α-rhamnoisorobin (2), afzelin (3), and
kaempferitrin (4) as pure compounds by far-infrared (FIR) irradiation of kenaf (Hibiscus
cannabinus L.) leaves. The depigmenting and anti-inflammatory activity of the compounds
was evaluated by analyzing their structure-activity relationships. The order of the
inhibitory activity with regard to depigmentation and nitric oxide (NO) production was
kaempferol (1) > α-rhamnoisorobin (2) > afzelin (3) > kaempferitrin (4). However, α-
rhamnoisorobin (2) was more potent than kaempferol (1) in NF-κB-mediated luciferase
assays. From these results, we conclude that the 3-hydroxyl group of kaempferol is an
important pharmacophore and that additional rhamnose moieties affect the biological
activity negatively.

Keywords: kaempferol; rhamnoside; depigmentation; anti-inflammation

Molecules 2011, 16 3339

1. Introduction

Flavonoids are naturally occurring phenolic phytochemicals which are reported to possess various
biologically important properties [1]. Consequently, the structure-activity relationships (SAR) of
flavonoids have received much attention [2]. Despite the similarities among the flavonoid structures,
minor structural modifications cause significant variations in their biological properties, with the
number and specific positions of the hydroxyl groups determining the type and intensity of their
activity. Generally, flavonoid glycosides are more abundant than flavonoids in natural products [3].
However, the SARs of flavonoid glycosides have not been fully studied [4]. Kenaf (Hibiscus
cannabinus L.) is a member of the Malvaceae family and is used as a traditional folk medicine in
Africa and India. It exhibits a broad spectrum of biological properties such as hepatoprotective activity
[5], anti-oxidative activity [6], haematinic activity [7], and immunomodulatory effects [8]. Recently,
we identified kaempferitrin (kaempferol-3,7-O-α-dirhamnoside) as one of the main components in
kenaf leaf extracts. Far-infrared (FIR) irradiation dry kenaf leaf powder showed derhamnosylation
products. Recently, the derhamnosylation products were isolated as pure compounds [9]. Chemical
investigations identified their structures as kaempferol, α-rhamnoisorobin (kaempferol 7-O-α-L-
rhamnopyranoside), and afzelin (kaempferol 3-O-α-L-rhamnopyranoside) (Figure 1). In this study, we
evaluated the depigmenting and anti-inflammatory properties of kaempferol and its rhamnosides by
analyzing their structure-activity relationships.

Figure 1. Structure of kaempferol and its rhamnosides.

OH OH
OH
HO O HO O O

O
OH HO OH
OH O Me OH O

Kaempf erol (1) α-Rhamnoisorobin (2)

OH OH
OH
HO O HO O O

O O Me O
HO O O Me
OH O Me OH O
HO OH HO OH
OH OH
Af zelin (3) Kaempf eritrin (4)

2. Results and Discussion

2.1. Depigmenting activity

We previously reported the anti-tyrosinase activities of kaempferol and its rhamnosides [9].
Compounds 1 (kaempferol) and 2 (α-rhamnoisorobin) showed tyrosinase inhibitory activities.
Molecules 2011, 16 3340

However, compounds 3 (afzelin) and 4 (kaempferitrin) showed no inhibitory activities. In this study,
we investigated the relationship of structure and biological activity in these compounds by evaluating
the inhibitory activity of kaempferol and its rhamnosides on melanogenesis and inflammation. The
depigmenting activity and cytotoxicity are summarized in Table 1.

Table 1. Depigmenting activities of kaempferol and its rhamnosides.

Inhibitory activitya [IC50, (μM)]
Compound
Tyrosinase Depigmentation Cytotoxicity
Arbutin 330.2 ± 1.1 170.8 ± 2.1 >200
Kaempferol (1) 171.4 ± 0.9 37.66 ± 0.1 25.6 ± 0.9
b
α-Rhamnoisorobin (2) >400 (39.1 %) 39.45 ± 0.4 22.9 ± 1.3
Afzelin (3) >400 >100 >100
Kaempferitrin (4) >400 >100 >100
a
Values were determined from logarithmic concentration-inhibition curves and are the means of
three experiments. b Inhibitory activity (% of control) at 400 μM.

We used B16 melanoma cells to evaluate the depigmenting activity [10]. Arbutin was used as the
positive control. Compounds 1 (kaempferol) and 2 (α-rhamnoisorobin) showed inhibitory activity.
However, their activity may originate from their cytotoxicity, as 1 and 2 showed cytotoxicity towards
B16 melanoma cells. Compounds 3 (afzelin) and 4 (kaempferitrin) showed neither inhibitory activity
nor cytotoxicity. These results suggest that the 3-hydroxyl group of kaemferol is a pharmacophore for
depigmenting activity and cytotoxicity.

2.2. Anti-inflammatory activity

Next, we evaluated the anti-inflammatory activity by measuring both the inhibition of the nitric
oxide (NO) production [11] and the suppression of the NF-κB-mediated luciferase assays [12]. We
estimated the inhibitory activity of kaempferol and its rhamnosides relative to NO production, induced
by LPS in RAW264.7 cells, and the NF-κB-mediated luciferase activity relative to a non-specific
phosphodiesterase inhibitor such as pentoxifylline and BAY11-7082, an IκBα kinase inhibitor, as
positive controls. Table 2 shows the inhibitory activity of kaempferol and its rhamnosides against NO
production.

Table 2. NO inhibitory activity of kaempferol and its rhamnosides.

Inhibitory activitya [IC50, (μM)]
Compound
NO Cytotoxicity
Pentoxifylline 446.0 ± 1.1 >1000
Kaempferol (1) 15.4 ± 0.2 >100
α-Rhamnoisorobin (2) 37.7 ± 2.0 >100
b
Afzelin (3) >100 (98.3 %) >100
b
Kaempferitrin (4) >100 (108.1 %) >100
a
Values were determined from logarithmic concentration-inhibition curves and are the means of
three experiments. b Inhibitory activity (% of control) at 100 μM.
Molecules 2011, 16 3341

The inhibitory activity for NO production is very similar to that of tyrosinase inhibition. The NO
inhibitory activity decreased in the order of kaempferol (1), α-rhamnoisorobin (2), afzelin (3), and
kaempferitrin (4). Compounds 1 (IC50 = 15.4 μM) and 2 (IC50 = 37.7 μM) showed potent inhibitory
activity without cytotoxicity. An additional rhamnose moiety at the 3-position caused a loss in NO
inhibitory activity. The effect of kaempferol and its glycosides on the NF-κB-mediated luciferase
activity was also evaluated (Table 3).

Table 3. Effects of kaempferol and its glycosides on the NF-κB-mediated luciferase activity.
Compound Inhibitory activitya [IC50, (μM)]
BAY11-7082 11.5 ± 0.1
Kaempferol (1) 90.3 ± 5.1
α-Rhamnoisorobin (2) 36.2 ± 3.3
Afzelin (3) >100
Kaempferitrin (4) >100
a
Values were determined from logarithmic concentration-inhibition curves and are the means of
three experiments.

Kaempferol (1) and α-rhamnoisorobin (2) also inhibited the NF-κB-mediated luciferase activities.
However, the activity order of the two compounds was different. Compound 2 (IC50 = 36.2 μM) was
more potent than compound 1 (IC50 = 90.3 μM).

2.3. Expression of iNOS mRNA

To elucidate the mechanism underlying the inhibition of the NO production, we examined the
effectof kaempferol (1) and α-rhamnoisorobin (2) on the expression of iNOS mRNA in LPS-activated
RAW 264.7 cells [13]. It is well known that iNOS facilitates the synthesis of NO, and the
transcriptional factor of NF-κB indirectly causes suppression of the NO production by affecting the
iNOS expression [14].
RAW 264.7 cells were treated with different concentrations of compound 1 and 2 (25–100 μM) and
then stimulated with LPS (1 μg/mL) for 6 h. The treatment with kaempferol (1) and α-rhamnoisorobin
(2) suppressed the expression of iNOS mRNA in a dose-dependent manner (Figure 2). These results
suggested that the inhibition of NO production by compound 1 and 2 was caused by the suppression of
the iNOS mRNA. The effect of kaempferol (1) on iNOS mRNA expression was more potent than that
of α-rhamnoisorobin (2).

3. Experimental

3.1. Measurements of melanin content

B16 cells (5 × 104 cells/well) were incubated with various concentrations of kaempferol and its
rhamnosides or arbutin for 5 days. The cell pellets were then dissolved in 1 N NaOH in 10% DMSO
(500 μL) at 80 °C for 1 h. The relative melanin content was determined by measuring the absorbance
Molecules 2011, 16 3342

at 475 nm with an ELISA reader (Molecular Devices Corp., Menlo Park, CA, USA). A standard
synthetic melanin curve (0–500 μg⁄mL) was prepared in triplicate for each experiment.

Figure 2. Effects of kaempferol (1) and α-rhamnoisorobin (2) on the expression of iNOS
mRNA in LPS-activated macrophages. **: p < 0.01 compared to control.

3.2. Measurements of NO production

RAW264.7 cells (1 × 106 cells/mL) were preincubated with kaempferol and its rhamnosides or
arbutin for 30 min and continuously activated with LPS (1 μg/mL) for 24 h. The nitrite in the culture
supernatants was measured by adding Griess reagent (100 μL, 1% sulfanilamide and 0.1% N-[1-
naphthyl]-ethylenediamine dihydrochloride in 5% phosphoric acid) to samples of the medium (100 μL)
for 10 min at room temperature. The OD at 570 nm (OD570) was measured using a Spectramax 250
microplate reader (Molecular Devices). A standard curve of NO was made with sodium nitrite.

3.3. Measurements of cytotoxicity

After the preincubation of RAW264.7 cells (1 × 106 cells/mL) for 18 h, kaempferol and its
rhamnosides or arbutin (0–100 μM) were added to the cells and incubated for 24 h. The cytotoxic
effect of kojyl thioether derivatives was then evaluated by a conventional MTT assay. At 3 h prior to
culture termination, MTT solution (10 μL, 5 mg/mL in a phosphate buffered-saline, pH 7.4) was added
and the cells were continuously cultured until termination. The incubation was halted by the addition
of 15% sodium dodecyl sulfate into each well, solubilizing formazan. The absorbance at
570 nm (OD570–630) was measured by a Spectramax 250 microplate reader.

3.4. Luciferase assay

HEK293 cells (1 × 106 cells/mL) were transfected with plasmids containing NF-κB-Luc, (1 μg/mL
each) as well as β-galactosidase (0.5 μg/mL) using the PEI method in a 12-well plate for 48 h, and
they were treated with testing compounds in the presence or absence of PMA (0.1 μM) for 24 h.
Luciferase assay was performed using the Luciferse Assay System (Promega).
Molecules 2011, 16 3343

3.5. mRNA detection by quantitative and semi-quantitative real-time reverse transcription-PCR

The total RNA from the LPS treated-RAW264.7 cells was prepared by adding TRIzol Reagent
(Gibco BRL), as per the manufacturer’s instructions. Real-time PCR reactions were conducted using
MuLV reverse transcriptase. The primers (Bioneer, Daejeon, Korea) were used as previously reported
[15].

3.6. Statistical analysis

Student’s t-test and one–way ANOVA were used to determine the statistical significance. Data
(Tables 1, 2, and 3, and Figure 2) expressed as means ± standard errors (SEM) were taken from at least
three independent experiments performed in triplicate. P < 0.05 was considered statistically significant.

4. Conclusions

In the present study, we have evaluated the biological activity of kaempferol (1) and some of its
rhamnosides such as α-rhamnoisorobin (2), afzelin (3), and kaempferitrin (4). Depigmenting and anti-
inflammatory activities such NO production and NF-κB-mediated luciferase activity were tested. In all
tested assays, kaempferol (1) and α-rhamnoisorobin (2) showed inhibitory activity. These results imply
that the 3-hydroxyl group of kaempferol is an important pharmacophore and that additional rhamnose
moieties at 3 of kaempferol have a negative effect on the biological activity. RT-PCR analysis
suggested that compound 1 and 2 inhibited the NO production by suppressing the iNOS mRNA
expression.

Acknowledgement

This work was supported by the small and medium business technology innovation development
project (S1075444) of the Korea small and medium business administration.

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Sample Availability: Samples of compounds1-4 are available from the authors

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